CN104725513A

CN104725513A - Fusion protein and its use in multiple sclerosis treatment

Info

Publication number: CN104725513A
Application number: CN201310713428.6A
Authority: CN
Inventors: 孙兵; 郑钰涵; 苏攀; 吴晓东
Original assignee: Shanghai Institutes for Biological Sciences SIBS of CAS
Current assignee: Shanghai Institutes for Biological Sciences SIBS of CAS
Priority date: 2013-12-20
Filing date: 2013-12-20
Publication date: 2015-06-24
Also published as: US20170022265A1; EP3085710A1; US10400027B2; EP3085710B1; WO2015090223A1; EP3085710A4

Abstract

The invention relates to a fusion protein and its use in multiple sclerosis treatment, and concretely relates to a fusion protein Fc-ECM1 of an extracellular stromatin 1 (ECM1), cloning, construction and expression thereof, and a use of the Fc-ECM1 protein in the preparation of medicinal compositions. The medicinal compositions are used for treating multiple sclerosis.

Description

Fusion rotein and the purposes in treatment multiple sclerosis thereof

Technical field

The invention belongs to biomedicine field and be specifically related to clone, the expression of fusion rotein, and medicinal use.This aspect also relates to the purposes of fusion rotein in treatment multiple sclerosis.

Background technology

multiple sclerosis and pathology thereof

Multiple sclerosis (multiple sclerosis, MS) be central nervous system (central nervous system, CNS) a kind of chronic auto-immune disease common in; pathology is main change with early stage alba demyelination and Quantifying axonal loss; cause various symptom; comprise sensation change, visual disorder, muscle weakness, melancholy, coordination and difficulty speaking, major fatigue, cognitive disorder, disequilibrium, body heat and pain etc., can cause reactivity obstacle and deformity time serious.

MS involves Chinese middle-aged adults more, has very high disability rate.Epidemiological study shows, the whole world 2,500,000 people that have an appointment suffer from multiple sclerosis, according to country or specific group different, morbidity is in every 100,000 people between 2 people to 150 people.The symptom of 80%MS patient belongs to recurrence-remission form (relapsing-remitting, RR), show as multiple relapse and alleviation, twice recurrence interval stable disease, along with the progressive deterioration of disease symptoms, after 10-20, about there is the RRMS patient of half can change secondary Advancement Type (secondary progressive, SP) into.Remaining 20%MS patient belongs to former and sends out progressive (primary progressive, PP), and after showing as morbidity, the state of an illness is without alleviation, in continuous progressive deterioration.

Large quantity research shows that panimmunity cell take part in the morbidity of MS, comprises T cell, B cell and scavenger cell etc.Experimental autoimmune encephalomyelitis (experimental autoimmune encephalomyelitis, EAE) central nervous system is occurred in, a kind of organ specific autoimmune's property disease mediated by activating T cell, be brought out by myelin protein antigen active immunity laboratory animal there is delayed allergy disease that is chronic, the flammatory demyelinating feature of repeated relapsing, CNS, major pathologic features be perivascular inflammatory cell invade profit and neural myelin react.EAE in mice (experimental autoimmune myelitis) model has identical feature in all many-sides such as clinical symptom, biochemical indicator, immunity and pathology with mankind MS, so it is current internationally recognized research MS ideal animals model (Chinese cytobiology journal, 34 volumes, 826-836 page).

Under normal physiological conditions, under hemato encephalic barrier (blood-brain barrier, BBB) controls, only there is the immunocyte of only a few can enter central nervous system and play immunosurveillance.But under virus/bacteriological infection or inflammatory conditions, a large amount of immunocytes of periphery can enter central nervous system CNS by hemato encephalic barrier.Inflammatory CD4 ⁺t cell invades CNS, make Activated Microglia, and the scavenger cell of recruiting inflammatory enters CNS, finally causes the myelin of CNS to be destroyed, less prominent shape glial cell death, the mechanism of EAE morbidity that Here it is.By suppressing inflammatory CD4 ⁺the migration of T cell, stops it to enter central nervous system, effectively can treat and alleviate EAE and multiple sclerosis.2010, Novartis Co., Ltd developed the oral pharmaceutical FTY720/Gilenya of first treatment multiple sclerosis, and its mechanism is exactly by suppressing CD4 ⁺multiple sclerosis symptom is alleviated in the migration of T cell.

existing medicine and deficiency thereof

The pathogenesis of MS is complicated, completely not clear and definite yet so far, does not also have special effective medicine to the treatment of MS.Change owing to there is significant inflammation in acute MS damage, Past 30 Years concentrates on anti-inflammatory aspect for the treatment emphasis of MS.

Existing 7 kinds of medicines go through for treatment patient MS at present, comprise glatiramer (glatiramer acetate, GA), recombinant interferon β, natalizumab (natalizumab), mitoxantrone (mitoxantrone) etc., these medicines are applicable to the MS of different symptoms separately, suitably can alleviate Relapsing Multiple Sclerosis, reduce the disease relapse of some patients or improve patient clinical symptom.Such as, but these medicines also also exist many side effects, and the common adverse reactions of recombinant interferon β is influenza-like symptom simultaneously, continue 24 ~ 48 hours, usually no longer occur after 2 ~ 3 months.IFN-β 1a can cause injection site red and swollen and pain, hepatic disorder and severe allergic reaction etc., IFN-β 1b can cause injection site red and swollen, touch a tender spot, occasionally cause local necrosis, serum transaminase slightly increase, oligoleukocythemia or anaemia.Natalizumab can cause progressive multifocal leukoencephalopathy (progressive multifocal leukoencephalopathy, PML).The common adverse effect of mitoxantrone comprises nauseating, baldness, oligoleukocythemia, anemia and myocardium toxicity etc.Therefore, how to develop new Diagnosis and Treat means for MS pathogenesis, research and development make new advances effectively and the little MS medicine of side effect, have very important scientific meaning.

extracellular CaM

Extracellular CaM (extracellular matrix protein1, ECM1) is the glycosylated protein of the 85KD size of being secreted by interstitial scleroblast system MN7, is found in 1994 at first.Researchist have found the ECM1 homologous gene being positioned at human chromosomal 1q21 position in 1997.Research subsequently shows, ECM1 is formed cartilage bone, and endotheli ocytosis and blood vessel occur all to play important regulating effect.From 2002, investigators find that ECM1 sudden change causes lipoid proteinosis (lipoid proteinosis) and can produce idiopathic ECM1 antibody in lichen sclerosis (lichen sclerosus) patient body successively, thus cause the forfeiture of ECM1 function and the generation etc. of disease, showing the critical function of ECM1 gene in dermatology, is also in recent years for the Main way of ECM1 research.But the expression level of wild-type ECM1 is low, not easily purifying, and may occur that in purge process protein structure changes, finally cause active loss.At present, for the rare report of the function of ECM1 in disease of immune system.

Summary of the invention

For the problems referred to above of wild-type ECM1, the present inventor expresses and functional study for the engineering of ECM1, constructs the fusion protein F c-ECM1 of ECM1, and research demonstrates the using value of engineering expression gained fusion rotein in treatment multiple sclerosis.

Contriver finds through research, Fc sequence in ECM1 albumen and human IgG is merged the albumen obtained and carry out purifying more easily by the mode of affinity chromatography, and suitable protein glycosylation modification can be obtained in host, thus the albumen expressed is made to keep the activity had and the function of wild-type protein.In the later stage of inducing mouse Autoimmune Encephalomyelitis (EAE) model, treat with Fc-ECM1 albumen, significantly can alleviate disease degree, show as disease scoring value and reduce, infiltration reduces to the inflammatory cell number of central nervous system, and pathology damage alleviates.Further investigation shows that Fc-ECM1 suppresses pathogenic T h cell to the migration of CNS.The medicine used for MS disease mostly at present is synthesis type, there is various different side effect, and ECM1 albumen is by Th2 cell-specific high expression level, its structure composition is the native protein that the mankind exist under physiological status, and we expect that it may be less to the side effect of human body.There is provided new theoretical foundation to the research of the Fc-ECM1 pathogeny that is EAE model and multiple sclerosis and treatment, Fc-ECM1 albumen has the potential value being developed further into treatment multiple sclerosis medicine.

A main purpose of the present invention be to provide a kind of can effectively engineering express Fc-ECM1 fusion rotein, this albumen is easy to purifying, and can maintain normal ECM1 protein function and activity.

In one embodiment, the invention provides the fusion protein F c-ECM1 of Fc sequence in extracellular CaM (ECM1) and human IgG.Particularly, in fusion rotein of the present invention, the aminoacid sequence of described extracellular CaM is as shown in the 41-580 amino acids of SEQ ID NO.2, and the aminoacid sequence of described Fc sequence is as shown in the 583-811 amino acids of SEQ ID NO.2.In a kind of embodiment, the sequence of described fusion rotein is as shown in the 41-811 amino acids of SEQ ID NO.2.

In another embodiment, the invention provides a kind of nucleic acid, it contains the coding nucleic acid of Fc-ECM1 fusion rotein.Particularly, described nucleic acid molecule contains the nucleotide sequence of encode either extracellular matrix albumen 1, such as but not limited to the 121-1740 position Nucleotide of SEQ ID NO.1, and the nucleotide sequence of described Fc sequence of encoding, such as but not limited to the 1747-2433 position Nucleotide of SEQ ID NO.1.Alternatively, described nucleic acid is also containing the coding nucleotide sequence of signal peptide sequence, and the coding nucleotide sequence of insect signal peptide shown in the 2-40 amino acids including but not limited to SEQ ID NO.2, such as but not limited to the 4-120 position Nucleotide of SEQ ID NO.1.In a kind of embodiment, the sequence of described nucleic acid is as shown in the 1-2436 position of SEQ ID NO.1,4-2436 position, 1-2433 position, 4-2433 position, 121-2436 position or 121-2433 position Nucleotide.

In another embodiment, the invention provides a kind of expression vector, in order to express Fc-ECM1 fusion rotein.In a kind of embodiment, described expression vector contains the coding nucleic acid of Fc-ECM1 fusion rotein.In another embodiment, the invention provides a kind of host cell of expressing Fc-ECM1 fusion rotein.In a kind of embodiment, described host cell is insect cell eukaryotic expression system, as insect baculovirus expression system.

A main purpose of the present invention is to provide a kind of pharmaceutical composition being used for the treatment of multiple sclerosis.In one embodiment, described pharmaceutical composition comprises the extracellular CaM that (A) treats significant quantity; And (B) acceptable vehicle or vehicle pharmaceutically or in immunology.In a kind of embodiment, in described pharmaceutical composition, extracellular CaM accounts for 0.001 ~ 99.9wt% of pharmaceutical composition gross weight.In a preferred embodiment, in described pharmaceutical composition, extracellular CaM accounts for 1 ~ 95wt% of pharmaceutical composition gross weight, is preferably 5 ~ 90wt%, more preferably 10 ~ 80wt%.In a kind of embodiment, described vehicle includes but not limited to water, aqueous buffer solution, ethanol, polyvalent alcohol, vegetables oil, injectable organic ester, and composition thereof.Particularly, described aqueous buffer solution includes but not limited to lower group: phosphate buffered saline buffer, Tris damping fluid, borate buffer solution, Succinate Buffer, histidine buffering liquid or citrate buffer.

Another main purpose of the present invention is to provide a kind of method for the treatment of multiple sclerosis, and described method comprises the object that the Fc-ECM1 fusion rotein for the treatment of significant quantity is given needs acquisition treatment or alleviates.Object can be given with the form of pharmaceutical composition by described Fc-ECM1 fusion rotein.Described object can be Mammals, such as people.Described method can be treated by the migration of suppressor T cell or alleviate multiple sclerosis.

Another main purpose of the present invention is to provide the purposes of extracellular CaM in medicine preparation, and described medicine is used for the treatment of or alleviates multiple sclerosis.

In some embodiments, pharmaceutical composition of the present invention or method can effectively be treated multiple sclerosis or alleviate its symptom, and described symptom includes but not limited to lower group: sensation change, visual disorder, muscle weakness, melancholy, coordination and difficulty speaking, major fatigue, cognitive disorder, disequilibrium, body heat, pain, reactivity obstacle and deformity etc.

In some embodiments, in pharmaceutical composition of the present invention or method, described Fc-ECM1 fusion rotein and water-based and non-aqueous carrier can be mixed for administration or pharmaceutical compositions.The pharmaceutically acceptable vehicle being used for the treatment of application is that pharmaceutical field is well-known.Described vehicle can include but not limited to: water, aqueous buffer solution, ethanol, polyvalent alcohol (as glycerine, propylene glycol, polyoxyethylene glycol etc.) and their suitable mixture, vegetables oil is as sweet oil, and injectable organic ester is as ethyl oleate etc.Described aqueous buffer solution includes but not limited to: phosphate buffered saline buffer, Tris damping fluid, borate buffer solution, Succinate Buffer, histidine buffering liquid or citrate buffer etc.

In some embodiments, extracellular CaM of the present invention can be given by oral, injection, local or be given object with other known technology.Described injection includes but not limited to: subcutaneous injection, intravenous injection, intraperitoneal, intramuscular injection, breastbone inner injection and infusion.The extracellular CaM of significant quantity can be given by single or or be repeatedly supplied to object, such as once a day, the next day once, three days once, twice weekly, once in a week, monthly twice, monthly etc.In some embodiments, the dosage of this extracellular CaM is about 60mg/kg to 0.025mg/kg, more preferably from about 0.025mg/kg to 15mg/kg, such as 4mg/kg or 5mg/kg.Those skilled in the art can adopt known technology to determine definite dosage and preparation according to the object for the treatment of and the result for the treatment of of required realization, see such as, Remington:The Science and Practice of Pharmacy (" Lei Mingdun: pharmaceutical science and put into practice "), Gennaro compiles (2003 the 20th edition), and Pickar, Dosage Calculations (" Rapid Dose Calculation ") (1999)) etc.

Based on disclosure herein, other side of the present invention will be apparent to those skilled in the art.

Brief Description Of Drawings

Fig. 1 shows the coomassie brilliant blue staining figure of purifying protein.

Fig. 2 is presented in mouse EAE model the progress giving Fc-ECM1 fusion rotein and can alleviate EAE disease.

Fig. 3 shows the migration of Fc-ECM1 suppressor T cell.

The immunne response level that Fc-ECM1 does not affect peripheral lymphoid organs is used in Fig. 4 display.

Fig. 5 shows Fc-ECM1 albumen does not affect Th17 cytodifferentiation and cell proliferation.

In each figure, error line represents standard error, and * represents p<0.05, and * * represents p<0.01.

Embodiment

Prove that Fc-ECM1 of the present invention is in the effect for the treatment of in multiple sclerosis and the mechanism of action thereof below by way of corresponding mouse model, the experimental evaluation provided only is not used in for exemplary illustration the present invention and limits the scope of the invention.The experimental technique of unreceipted actual conditions in embodiment, usually conveniently condition, or according to the condition that manufacturer advises.

I. materials and methods

experiment material

Animal

C57BL/6 mouse, purchased from Chinese Academy of Sciences's Shanghai Experimental Animal Center, is raised in Shanghai OEG cell institute of Chinese Academy of Sciences Experimental Animal Center (SPF level).

Main agents

Be derived from the Toxins, pertussis (PTX) of Bordetella pertussis (bordetella pertussis), and complete Freund's adjuvant (Freund ' s Adjuvant, Complete), Solvent Blue38 and Percoll is purchased from Sigma.DMEM, RPMI1640 substratum and foetal calf serum are purchased from Gibco BRL.Transfection reagent Lipofectamine is Invitrogen Products.Human IgG is biological purchased from neck tide.Immunocyte capable of blocking passes the micromolecular inhibitor GM6001 of hemato encephalic barrier basilar membrane purchased from Millipore.

MOG _35-55: MEVGWYRSPFSRVVHLYRNGK, by biosynthesizing of shining by force.

Unless expressly stated otherwise, all the other reagent are all purchased from Sigma.

Cytokine and antibody

In vitro T cell cultivate in, recombined small-mouse IL-12, IL-23 and human IL-2 purchased from R & D, recombined small-mouse IL-1 α, IL-6 and people TGF-β 1 purchased from Peprotech.Anti-mouse CD3 (145-2C11), anti-CD28 (37.51), anti-mouse IL-4 (11B11) purchased from BD Pharmingen, anti-mouse IFN-γ (XMG1.2) purchased from eBioscience.Cell sorting mouse CD4 antibody used is purchased from Miltenyi Biotec.

In FCM analysis experiment, FITC anti-mouse IFN-γ (XMG1.2) comes from BD Pharmingen purchased from eBioscience, FITC anti-mouse CD4 (GK1.5), PerCP anti-mouse CD4 (RM4-5), PE anti-mouse IL-17 (TC11-18H10).

In western blotting detects, HA mouse source monoclonal antibody (16B2) is Covance Products, Cat.#CO-MMS-101R.ECM1 polyclonal antibody is the preparation of this laboratory, we express the protokaryon albumen comprising mouse ECM1 gene mRNA 1024-1798 258, position amino acid and 282 amino acid fragments in 178-1024 position respectively, then with protokaryon protein immunization rabbit with obtain for N end and C hold resist more.The anti-rabbit IgG bis-of HRP mark resists for Southern Biotechnology Associates Products, and the anti-mouse IgG bis-of Cat.#4050-05, HRP mark resists for R & D Products, Cat.#HAF007.Except as otherwise noted, other reagent used in each embodiment and experiment equipment and equipment are medical grade.

the induction of EAE and marking

Prepare before experiment

A. the preparation of complete Freund's adjuvant (CFA): add heat-inactivated mycobacterium tuberculosis (TB) to final concentration 5mg/ml in incomplete Freund's adjuvant (IFA), fully mixing before using.

B. the emulsification of antigen: connect two glass needle tubings with Y-tube, PBS:CFA and antigen MOG (20mg/ml) to be added respectively in a needle tubing that (every 200 μ l emulsions are containing 300 μ g MOG, 85 μ l PBS and 100 μ l CFA), in eliminating needle tubing after bubble, promote needle tubing about 500 times back and forth, make each composition in needle tubing fully mix emulsification.The antigen that the immunity of C57BL/6 mouse adopts is MOG _35-55peptide section.MOG _35-55peptide section has and strong causes brain inflammatory, and can produce significant t cell immune response, be antigen peptide section most widely used in the induction of C57BL/6 mouse EAE model.

The foundation of EAE model and administration

A. the C57BL/6 female mice in 6-8 age in week is used in experiment, heavily about 25g.In immunity the same day (the 0th day), every mouse accepts antigen injection, point 3 adequately emulsified antigens of subcutaneous injection 200 μ l: hind leg thigh root, each 50 μ l in both sides, root of the tail portion 100 μ l; Only (being dissolved in 100 μ l PBS) give Toxins, pertussis (PTX) 300ng/ through abdominal injection simultaneously.

B. the 2nd day, again every mouse 300ng PTX (concentration is the same) is given through abdominal injection.

C. process is tested: at the 8th, 11 and 14 day of immunity, treatment group mouse Fc-ECM1 fusion rotein (100 μ g/ is given through tail vein injection, be dissolved in 200 μ l PBS), give the contrast human IgG albumen (100 μ g/ only, are dissolved in 200 μ l PBS) of control group mice respective volume.

EAE clinical score standard

From the immune same day, the clinical manifestation of observation experiment mouse, experimentally required carry out EAE disease score and record corresponding score value.Scoring continues 20 days to 60 days, is generally about 30 days, if experiment mice is because of disease death, and termination of giving a mark.Standards of grading are as follows,

0 point: performance without exception;

1 point: afterbody is paralysed;

2 points: hind leg is got involved, unable, walk lamely;

3 points: hind leg is paralysed completely;

4 points: two hind limb paralysis forelimb are got involved;

5 points: dead.

histopathologic slide dyes

Paraffin embedding mouse spinal cord tissue

A. tissue sampling is with fixing: obtain mouse spinal cord sample at EAE in mice onset peak period.Specific as follows: within the 24th day after EAE immunity, by mouse anesthesia, after 4% paraformaldehyde perfusion, to be separated and to obtain mouse spinal cord sample, then fix 2-3 days by 4% paraformaldehyde room temperature.

B. serial dehydration and transparent: the spinal cord marrow fixed is placed successively in 50%, 70%, 80%, 95%, 100% ethanolic soln and makes it dehydration for each 1 hour.Then by spinal cord at ethanol: dimethylbenzene ratio is place 1 hour in the dimethylamino ethanol benzole soln of 1:1, places 5-10 minute after taking-up in dimethylbenzene, treats that tissue is close to till transparent.Put into paraffin 60 DEG C of waxdips to spend the night.

C. embed: after spinal cord being cut into the fritter of about 0.3cm, working specification carries out paraffin embedding routinely, and-20 DEG C save backup.

HE dyes

Taken out by-20 DEG C of paraffin sections preserved, after 37 DEG C of oven dry, dewax successively by standard method, rehydration, brazilwood extract dyeing, eosin stains, dewaters transparent, mounting, then dry for standby.

Fast Blue dyes

-20 DEG C of paraffin sections preserved are taken out, 37 DEG C dry then dewaxing and rehydration after, the FastBlue solution 60 DEG C being placed in 0.1% spends the night, clear water washes away loose colour, differentiation, repeats differentiation to basis of microscopic observation spinal cord periphery white matter for blue and inner grey matter is colourless, eosin stains, violet staining, dewaters transparent, and mounting post-drying is for subsequent use.

the preparation of Fc-ECM1 fusion rotein

For obtaining that there is bioactive Fc-ECM1 fusion rotein, utilize insect cell eukaryotic expression system (Bac-to-Bac Baculovirus Expression Systems, purchased from Invitrogen company) adopt the encoding gene of ECM1 to build nucleotide sequence as shown in SEQ ID No.1, in order to express the ECM1 albumen (Fc-ECM1 fusion rotein, aminoacid sequence is as shown in SEQ ID No.2) merged with human IgG Fc peptide section.

SEQ ID No.1 (coding nucleotide sequence of Fc-Ecm1 albumen):

ATG GCCTCTGAGGGAGCCTTCAAGGCTTCAGACCAGCGAGAGATGACGCCAGAGCGCCTCTTCCAGCACCTCCATGAAGTAGG TTATGCAGCACCCCCTTCCCCACCACAAACCCGGAGACTCCGAGTTGACCACTCTGTAACTTCTCTGCATGACCCTCCCCTCTTTGAGGAACAAAGAGAAGTGCAGCCCCCTTCCTCTCCAGAAGACATCCCTGTGTACGAGGAAGACTGGCCCACTTTCCTAAACCCTAATGTAGATAAAGCTGGTCCTGCTGTCCCTCAAGAAGCCATCCCCCTGCAGAAAGAGCAGCCCCCTCCCCAAGTCCATATTGAACAGAAGGAAATAGACCCGCCTGCCCAGCCTCAGGAGGAGATTGTCCAGAAAGAGGTGAAGCCACACACCTTGGCGGGCCAGCTCCCTCCAGAGCCCCGGACTTGGAATCCAGCCCGTCACTGCCAGCAGGGACGGAGAGGTGTCTGGGGCCACCGGCTGGATGGCTTCCCTCCTGGACGGCCTTCTCCAGACAATCTGAAGCAGATCTGCCTTCCTGAGCGTCAGCATGTGATCTACGGCCCCTGGAACCTGCCGCAGACTGGCTACTCTCACCTTAGTCGCCAGGGAGAGACCCTCAATGTGCTGGAGACCGGATACTCCCGCTGCTGTCGCTGCCGCAGCGACACAAACCGCCTAGACTGTTTGAAGCTTGTGTGGGAGGATGCAATGACCCAATTTTGTGAGGCCGAATTCTCTGTCAAGACCCGCCCCCACCTGTGCTGCAGACTGCGTGGGGAGGAGCGATTCTCTTGCTTCCAGAAGGAAGCTCCTCGCCCAGACTACCTGCTCCGACCCTGCCCCGTCCACCAGAATGGCATGTCCTCAGGGCCCCAGTTGCCTTTCCCCCCGGGGTTGCCCACACCGGACAATGTCAAAAACATCTGTCTCCTGAGACGCTTCCGCGCCGTGCCACGCAACCTCCCAGCTACTGACGCCATCCAGAGGCAGCTGCAGGCTCTGACTCGGCTGGAGACGGAGTTCCAGCGCTGCTGCCGCCAGGGCCACAACCACACTTGCACATGGAAGGCCTGGGAGGGTACCCTGGATGGATACTGCGAGCGGGAGCTGGCTATAAAGACCCACCCCCACTCGTGCTGCCACTACCCTCCTAGTCCTGCCCGTGATGAGTGCTTCGCCCACCTAGCTCCCTATCCCAACTATGACCGGGATATCTTGACCCTTGACCTCAGCCGAGTCACCCCCAACCTCATGGGCCAGCTCTGTGGAAGTGGAAGGGTCCTTAGCAAGCATAAACAGATTCCGGGGCTGATCCAGAATATGACCATCCGCTGCTGCGAGCTTCCATATCCAGAACAGGCCTGCTGCGGCGAAGAGGAGAAACTGGCCTTCATCGAGAACCTCTGTGGTCCCCGGAGGAATTCGTGGAAAGACCCTGCCCTCTGCTGTGACCTGTCTCCTGAAGATAAGCAAATCAACTGCTTCAATACCAACTACCTGAGGAACGTGGCTTTAGTGGCTGGAGACACTGGGAATGCCACTGGCTTGGGGGAGCAGGGCCCAACTCGGGGAACAGATGCCAACCCCGCCCCTGGGTCCAAGGAAGAA ACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAAAAGCTTGTCGAGAAGTACTAG

Wherein, initial 3 Nucleotide are initiator codon, 3, end Nucleotide is terminator codon, bolded section coding insect signal peptide sequence, band underscore code segment Fc-tag sequence, the ctcgag sequence represented with bold case lower case letters is manually-injected restriction enzyme site, and rest part is the encoding sequence of ECM1 albumen.

SEQ ID No.2 (aminoacid sequence of the Fc-Ecm1 albumen of band signal peptide):

ASEGAFKASDQREMTPERLFQHLHEVGYAAPPSPPQTRRLRVDHSVTSLHDPPLFEEQREVQPPSSPEDIPVYEEDWPTFLNPNVDKAGPAVPQEAIPLQKEQPPPQVHIEQKEIDPPAQPQEEIVQKEVKPHTLAGQLPPEPRTWNPARHCQQGRRGVWGHRLDGFPPGRPSPDNLKQICLPERQHVIYGPWNLPQTGYSHLSRQGETLNVLETGYSRCCRCRSDTNRLDCLKLVWEDAMTQFCEAEFSVKTRPHLCCRLRGEERFSCFQKEAPRPDYLLRPCPVHQNGMSSGPQLPFPPGLPTPDNVKNICLLRRFRAVPRNLPATDAIQRQLQALTRLETEFQRCCRQGHNHTCTWKAWEGTLDGYCERELAIKTHPHSCCHYPPSPARDECFAHLAPYPNYDRDILTLDLSRVTPNLMGQLCGSGRVLSKHKQIPGLIQNMTIRCCELPYPEQACCGEEEKLAFIENLCGPRRNSWKDPALCCDLSPEDKQINCFNTNYLRNVALVAGDTGNATGLGEQGPTRGTDANPAPGSKEE TCPPCPAPELLGGPSVPLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKKLVEKY

Wherein, bolded section is the insect signal peptide of methionine(Met) coded by initiator codon, band underscore part is Fc-tag aminoacid sequence, and the amino acid of the le sequence represented with bold case lower case letters coded by artificial restriction enzyme site, all the other are ECM1 protein amino acid sequence.

Basic procedure is as follows: by the encoding gene access pFastBac of nucleotide sequence as shown in SEQ ID No.2 ^tMin carrier, screening obtains baculovirus shuttle vector (Bacmid) DNA of correct restructuring, then adherent insect cell line HighFive is transfected into CellFECTIN, the viral supernatants obtained through coomassie brilliant blue staining and western blotting checking errorless after carry out protein expression in order to infect suspension type HighFive, obtain target protein (purifying protein coomassie brilliant blue staining figure as shown in Figure 1) by protein purification after results albumen supernatant, can be used for cell and experimentation on animals.Agents useful for same is all purchased from Invitrogen company.The Fc-ECM1 fusion rotein (sequence is as shown in the 41-811 amino acids of SEQ ID No.2) all using this section to prepare in active testing and function test.

cell purification and vitro differentiation

CD4+T cell and CD11c+DC cell obtain (Miltenyi Biotec) by magnetic bead sorting.Positive cell purity is usually above 95%.In T cell vitro differentiation system, T cell is incubated at and is added with 10% foetal calf serum (GIBCO), glutamine (2mM), β-ME (50mM), penicillin (50 units per ml), Streptomycin sulphate (50mg/ml), in the RPMI1640 nutrient solution of Sodium.alpha.-ketopropionate (1mM) and Hepes (100mM).

T cell stimulates via the 5 anti-CD3 of μ g/ml wrapper sheet and the anti-CD28 of 2 μ g/ml solubilities, breaks up in following induced environment towards different directions:

Th0------50U/ml IL-2,10 μ g/ml anti-IFN-γ and the anti-IL-4 of 10 μ g/ml

Th1------10ng/ml IL-12,10 μ g/ml anti-IL-4 and 50U/ml IL-2

Th17-----1ng/ml TGF-β 1,20ng/ml IL-6,10ng/ml IL-1 α, 10ng/mlIL-23,10 μ g/ml anti-IFN-γ and the anti-IL-4 of 10 μ g/ml.

Experimentally demand, adds Fc-ECM1 albumen or contrast IgG (detailed in Example 4) of 20 μ g/ml concentration in induced environment.

matrigel invasion experiment (Matrigel invasion assay)

The cell of vitro differentiation described above added corresponding albumen or GM6001 at the 3rd day, and within the 4th day, collecting cell counting uses in order to Matrigel.If pathogenic T h cell, taken out by the inguinal lymph nodes of the 8th day mouse after EAE immunity, the monokaryon lymphocyte that separation obtains is with 3 × 10 ⁶/ ml cell density remises sharp 3 days with the MOG of 50 μ g/ml and counts afterwards in order to Matrigel use.

BD BioCoat ^tMmatrigel ^tMinvasion Chamber and Control Inserts all buys from BD Biosciences company, carries out cell invasion experimental implementation by raw manufacturer methods involving handbook.Matrigel (Matrigel) can imitate the basilar membrane in hemato encephalic barrier, and therapeutic T cell needs to arrive lesions position through basilar membrane, and Control Inserts covers without matrigel, and cell can directly be bored a hole.

The concise and to the point flow process of cell invasion experiment is as follows: be placed in Matrigel hole in 24 orifice plates, and in 37oC incubator, hydration 2 hours in serum-free RPMI1640, makes its activity recovery, then carefully remove RPMI1640.Cell is all with 1 × 10 ⁶the number of/ml is resuspended in serum-free RPMI1640, adds various albumen or inhibitor on request, and on Matrigel hole, room adds 500 μ l cells, and lower room adds the RPMI1640 of 500 μ l5% serum, and Control Inserts is the same.Place in 37oC incubator after 22 hours, collection moves to the cell of lower room and counts.The experimentally ratio calculation cell migration rate of group and control group, that is: cell count × 100% moved in the cell count of moving in mobility=Invasion Chamber hole/Control Inserts hole.

the preparation of mouse CNS mononuclear cell suspension

A. open thoracic cavity after experiment mice anesthesia, after heart perfusion PBS, take out spinal cord.

B. gained marrow is inserted 70 μm of cell strainer, use plunger gentle abrasion, PBS rinses collection organization's homogenate.

C.2000rpm after centrifugal 5 minutes, abandon supernatant, the resuspended one-tenth homogenate of agglomerate will be organized with 7ml30%Percoll (Sigma), and then 4ml70%Percoll liquid slowly be added bottom tissue suspension and (insert bottom centrifuge tube with dropper), centrifugal 22 minutes of 2000rpm.

D. collect the monocyte being positioned at 70% and 30%Percoll liquid level middle layer, train liquid with the RPMI1640 containing 2%FBS and wash one time, then be resuspended in appropriate training in liquid, count stand-by.

fCM analysis

Infiltrate to CD4 in CNS monocyte to detect ⁺t cell ratio, the antibody marking anti-mouse CD4 with PE dyes.Cell carries out analyzing (BD Biosciences company) via FACSCalibur flow cytometer.

eLISA tests

In order to detect the cytokine concentration in cells and supernatant, buying IL-17 and IFN-γ ELISA dual-reagent box from R & D Systems and operating according to manufacturer's methods involving handbook.

³ h proliferation experiment

Be dissolved in PBS by anti-CD 3 antibodies with the concentration of 1 μ g/ml, by 60 μ g/ hole bags by 96 orifice plates, 4 DEG C are spent the night, and exhaust the supernatant in each hole before inoculating cell.Stimulate the CD4 of purifying with anti-CD 3 antibodies in 96 pre-coated orifice plates ⁺t cell, every hole is inoculated into 2 × 10 ⁵cell, with 200 μ l nutrient solutions cultivate.Growth of Cells is after 60 hours, every hole add 1 μ Ci [ ³h] thymus pyrimidine, after 12 hours by detect [ ³h] mixing of thymus pyrimidine and reflect the proliferative conditions of cell.Each three the multiple holes of each sample.Cultivation terminate rear cell collector collecting cell and with scintillation counter detect [ ³h] incorporation of thymus pyrimidine.

II. embodiment 1: later stage Fc-ECM1 protein for treatment effectively alleviates the EAE symptom in EAE mouse model

We select the C57BL/6 female mice in 6-8 week to carry out inducing mouse EAE model, often organize each 9, random assignment, the 0th day subcutaneous inoculation MOG _35-55, the 0th and 2 days abdominal injection PTX, part that detailed process sees above " foundation of EAE model and administration ".Mouse Fc-ECM1 albumen is given at selected time point.Use MOG _35-55the C57 mouse that antigen carries out EAE immunity is divided into 2 experimental group, Fc-ECM1 albumen or contrast IgG albumen (being only at every turn 100 μ g/) is given by tail vein injection respectively at the 8th, 11 and 14 day, day by day the clinical marking of EAE is carried out, to study the impact of Fc-ECM1 albumen on EAE disease.What Fig. 2 A showed is EAE marking situation, the EAE of Fc-ECM1 protein for treatment group mouse falls ill and is starkly lower than reference protein group, from the 21st day, the disease scoring difference between protein for treatment group and reference protein group had significant difference (p value <0.05).

Latter 24th day of immunity, obtain disease mouse spinal cord, Percoll is separated mononuclearcell, and with PE-anti-CD4 antibody surface mark after counting, FACS detection CD4+T cell proportion, calculates CD4+T cell number.After Fig. 2 B is presented at Fc-ECM1 protein for treatment, infiltration significantly reduces to the cd4 cell number of CNS.Fig. 2 C and D is the mouse spinal cord staining pathologic section result of falling ill obtained for after EAE immunity the 24th day.Fig. 2 C is HE dyeing, show that Fc-ECM1 treatment group mouse infiltrates the inflammatory cell number minimizing of CNS equally, Fig. 2 D is that Luxol Fast blue dyes, and shows that the demyelinating disease apparition of Fc-ECM1 treatment group mouse is less than control group mice (in figure, arrow indication is demyelinated sites).Fig. 2 mainly illustrates to carry out treating with Fc-ECM1 albumen to the CNS phase of moving at EAE model pathogenic T cell significantly can alleviate the severity of disease.Defining of described migration phase can see the record of pertinent literature, as " EMMPRIN:A Novel Regulator of Leukocyte Transmigration into the CNS in Multiple Sclerosis and Experimental Autoimmune Encephalomyelitis ", The Journal of Neuroscience, January12,201131 (2): 669 – 677.

III. the migration of embodiment 2:Fc-ECM1 suppressor T cell

Inventors have investigated the impact of Fc-ECM1 albumen on Th cell migration.Matrigel can imitate the basilar membrane in hemato encephalic barrier, and Th cell needs could to arrive lesions position through basilar membrane and plays function.We have selected the transfer ability that matrigel invasion experiment (Matrigel Invasion Assay) detects Th cell.Separating mouse CD4+T cell, stimulate activation ripe via the 5 anti-CD3 of μ g/ml wrapper sheet and the anti-CD28 of 2 μ g/ml solubilities, respectively organize when Th cell cultures the 3rd day and take respectively not process, add reference protein human IgG, Fc-ECM1 albumen or GM6001, the cell cultivated the 4th day carries out matrigel invasion experiment.Matrigel invasion experiment shows that the migration of Fc-ECM1 albumen to Th cell has restraining effect (Fig. 3 A, * represents p<0.05).

Since experiment in vitro has proved that Fc-ECM1 albumen can suppress Th cell migration, contriver have detected the impact of Fc-ECM1 albumen on pathogenic T cell migration ability with matrigel invasion subsequently.The inguinal lymph node cells of the 8th day mouse after EAE immunity takes out by we, and MOG remises the sharp cell obtained for 3 days and is used for doing Cell migration assay.Consistent with Vitro Experimental Results, corresponding contrast IgG is added respectively in matrigel invasion experiment, Fc-ECM1 or GM6001, ECM1 significantly can suppress the migration of pathogenic T cell, quite (Fig. 3 B, * represent p<0.05 (albumen consumption is the same) for inhibiting rate and GM6001.Above-mentioned experimental result illustrates, Fc-ECM1 albumen can suppress the migration of pathogenic T cell.Shown in Fig. 2 B, after ECM1 protein for treatment, infiltration significantly reduces to the cd4 cell number of CNS, illustrates that Fc-ECM1 may be migrate to caused by central nervous system through hemato encephalic barrier owing to which inhibits pathogenic cd4 cell to the therapeutic action of EAE.

IV. embodiment 3: later stage Fc-ECM1 protein for treatment does not affect immunne response and the cell proliferation level of peripheral lymphoid organs

The research of the influence factor of EAE disease mainly concentrates on two aspects, and one is the generation of antigen-specific pathogenic T cell, and two is the migrations to CNS system of pathogenic T cell.When first contriver have detected EAE immunity used in embodiment 3 and protein for treatment, the immunne response level of peripheral lymphoid organs.Get the immunity mouse spleen of the 15th day and lymphoglandula, separating monocytic cell, after remising sharp 3 days with 50 μ g/mlMOG, detect cytokine secretion in supernatant.As shown in Figure 4 A, no matter be spleen or lymphoglandula, MOG is external remises the equal no significant difference of expression level swashing rear IL-17 and IFNg between Fc-ECM1 treatment group and reference protein group.

Contriver also passes through simultaneously ³h incorporation methods have detected the heavy post-stimulatory proliferative conditions of two groups of Mouse spleen cells MOG.Separating monocytic cell described above and with the heavy irritation cell of MOG add after 60 hours [ ³h] thymus pyrimidine, within the 72nd hour, detect [ ³h] incorporation of thymus pyrimidine, do not observe significant difference (Fig. 4 B) equally.This illustrates that Fc-ECM1 albumen does not affect the generation of periphery pathogenic T cell in EAE disease model.

V. embodiment 4:Fc-ECM1 albumen does not affect the vitro differentiation of Th1 and Th17 cell

Think that Th1 and Th17 cell is main pathogenic T h cell at present, therefore contriver finally detects and add the impact of Fc-ECM1 albumen on differentiation in CD4+T cells in vitro differentiation Th1 and Th17 system.Obtain mouse CD4+T cell by Beads enrichment, press Th1 and Th17 differentiation condition respectively and cultivate, add Fc-ECM1 albumen or reference protein, break up after 4 days in culture system, collecting cell is used for dyeing in born of the same parents and carries out flow cytometer detection, and supernatant is used for ELISA and detects.IFN-γ and IL-17 is respectively generally acknowledged Th1 cell and the Th17 cell sign sexual cell factor, FACS(Fig. 5 A) and ELISA(Fig. 5 B) result all shows, adds the vitro differentiation that Fc-ECM1 albumen does not affect Th1 and Th17 cell.

Below in conjunction with specific embodiments, the clone of Fc-ECM1 albumen, expression and purification has been set forth, and the purposes in treatment multiple sclerosis.Should be understood that these embodiments are only not used in for illustration of the present invention to limit the scope of the invention.Those skilled in the art can make suitable amendment, variation to the present invention, understand version and the equivalent processes thereof of the method for application of physiologically active protein, and these amendments, change and equivalent processes are all within the scope of the present invention.

Reference

All documents that the present invention mentions are included in all by reference of text herein for all objects.

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2.Gasperini,C.,and Ruggieri,S.(2012).Development of oral agent in the treatment of multiple sclerosis:how the first available oral therapy,fingolimod will change therapeutic paradigm approach.Drug Des Devel Ther6,175-186.

3.Bhalerao,J.,P.Tylzanowski,J.D.Filie,C.A.Kozak,and J.Merregaert.(1995).Molecular cloning,characterization,and genetic mapping of the cDNA coding for a novel secretory protein of mouse.Demonstration of alternative splicing in skin and cartilage.The Journal of biological chemistry270:16385-16394.

4.Mongiat,M.,J.Fu,R.Oldershaw,R.Greenhalgh,A.M.Gown,and R.V.Iozzo.(2003).Perlecan protein core interacts with extracellular matrix protein1(ECM1),a glycoprotein involved in bone formation and angiogenesis.The Journal of biological chemistry278:17491-17499.

5.Smits,P.,Y.Poumay,M.Karperien,P.Tylzanowski,J.Wauters,D.Huylebroeck,M.Ponec,and J.Merregaert.(2000).Differentiation-dependent alternative splicing and expression of the extracellular matrix protein1gene in human keratinocytes.The Journal of investigative dermatology114:718-724.

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Claims

1. the fusion rotein of Fc sequence in an extracellular CaM and human IgG, the aminoacid sequence of described extracellular CaM is as shown in the 41-580 amino acids of SEQ ID NO.2, the aminoacid sequence of described Fc sequence is as shown in the 583-811 amino acids of SEQ ID NO.2, and the sequence of preferred described fusion rotein is as shown in the 41-811 amino acids of SEQ ID NO.2.

2. a nucleic acid, it contains the coding nucleic acid of fusion rotein described in claim 1, the encoding sequence of preferred described extracellular CaM is the 121-1740 position Nucleotide of SEQ ID NO.1, the encoding sequence of described Fc sequence is the 1747-2433 position Nucleotide of SEQ ID NO.1, described coding nucleic acid is optional comprises signal coding sequence, as the 4-120 position Nucleotide of SEQ ID NO.1; More preferably the sequence of described nucleic acid is as shown in the 1-2436 position of SEQ ID NO.1,4-2436 position, 1-2433 position, 4-2433 position, 121-2436 position or 121-2433 position Nucleotide.

3. express an expression vector for fusion rotein described in claim 1, preferred described expression vector is containing, for example nucleic acid according to claim 2.

4. a host cell, it comprises expression vector according to claim 3, and preferred described host cell is insect cell eukaryotic expression system.

5. a pharmaceutical composition, described pharmaceutical composition is used for the treatment of or alleviates multiple sclerosis, and described pharmaceutical composition comprises: (A) treats the fusion rotein according to claim 1 of significant quantity; And the acceptable vehicle of (B) pharmacy or vehicle.

6. pharmaceutical composition as claimed in claim 5, it is characterized in that, described fusion rotein accounts for 0.001 ~ 99.9wt% of described pharmaceutical composition gross weight, and/or described vehicle is selected from lower group of water, aqueous buffer solution, ethanol, polyvalent alcohol, vegetables oil, injectable organic ester, and composition thereof.

7. pharmaceutical composition as claimed in claim 6, it is characterized in that, described aqueous buffer solution is selected from lower group: phosphate buffered saline buffer, Tris damping fluid, borate buffer solution, Succinate Buffer, histidine buffering liquid, citrate buffer and composition thereof, and/or described polyvalent alcohol is selected from glycerine, propylene glycol, polyoxyethylene glycol and composition thereof, and/or described vegetables oil is sweet oil, and/or described injectable organic ester is ethyl oleate.

8. the purposes of fusion rotein according to claim 1 in medicine preparation, it is characterized in that, described medicine is used for the treatment of or alleviates the symptom of multiple sclerosis.

9. purposes as claimed in claim 8, it is characterized in that, described pharmaceutical pack contains: (A) treats the fusion rotein according to claim 1 of significant quantity; And the acceptable vehicle of (B) pharmacy.

10. purposes as claimed in claim 8, it is characterized in that, described Pharmaceutical formulations gives for oral, injection, local, and/or described Pharmaceutical formulations becomes presented in unit dosage form, preferred described injection is selected from lower group: subcutaneous injection, intravenous injection, intraperitoneal, intramuscular injection, breastbone inner injection and infusion.